Isolation and characterization of a cDNA encoding a mammalian cathepsin L-like cysteine proteinase from Acanthamoeba healyi

Abstract

We have cloned a cDNA encoding a cysteine proteinase of the Acanthamoebahealyi OC-3A strain isolated from the brain of a granulomatous amoebic encephalitispatient. A DNA probe for an A. healyi cDNA library screening was amplified by PCR usingdegenerate oligonucleotide primers designed on the basis of conserved amino acidsfranking the active sites of cysteine and asparagine residues that are conserved in theeukaryotic cysteine proteinases. Cysteine proteinase gene of A. healyi (AhCP1) wascomposed of 330 amino acids with signal sequence, a proposed pro-domain and apredicted active site made up of the catalytic residues, Cys25, His159 , and Asn 175 . Deducedamino acid sequence analysis indicates that AhCP1 belong to ERFNIN subfamily of C1peptidases. By Northern blot analysis, no direct correlation was observed between AhCP1mRNA expression and virulence of Acanthamoeba , but the gene was expressed at higherlevel in amoebae isolated from soil than amoeba from clinical samples. These findingsraise the possibility that Ahcp1 protein may play a role in protein metabolism anddigestion of phagocytosed bacteria or host tissue debris rather than in invasion ofamoebae into host tissue.